The present invention relates to novel phenol oxidizing enzymes, in particular, novel phenol oxidizing enzymes derived from strains of Stachybotrys and novel strains of the genus Stachybotrys producing these enzymes. The present invention provides methods and host cells for expressing Stachybotrys phenol oxidizing enzymes as well as methods for producing expression systems.
Phenol oxidizing enzymes function by catalyzing redox reactions, i.e., the transfer of electrons from an electron donor (usually a phenolic compound) to molecular oxygen (which acts as an electron acceptor) which is reduced to H2O. While being capable of using a wide variety of different phenolic compounds as electron donors, phenol oxidizing enzymes are very specific for molecular oxygen as the electron acceptor.
Phenol oxidizing enzymes can be utilized for a wide variety of applications, including the detergent industry, the paper and pulp industry, the textile industry and the food industry. In the detergent industry, phenol oxidizing enzymes have been used for preventing the transfer of dyes in solution from one textile to another during detergent washing, an application commonly referred to as dye transfer inhibition.
Most phenol oxidizing enzymes exhibit pH optima in the acidic pH range while being inactive in neutral or alkaline pHs.
Phenol oxidizing enzymes are known to be produced by a wide variety of fungi, including species of the genii Aspergillus, Neurospora, Podospora, Botytis, Pleurotus, Fomes, Phlebia, Trametes, Polyporus, Rhizoctonia and Lentinus. However, there remains a need to identify and isolate phenol oxidizing enzymes, and organisms capable of naturally-producing phenol oxidizing enzymes, which present pH optima in the alkaline range for use in detergent washing methods and compositions.
The present invention relates to novel phenol oxidizing enzymes obtainable from Stachybotrys. In particular, the enzymes of the present invention are capable of modifying the color associated with dyes and colored compounds having different chemical structures, especially at neutral or alkaline pH. Based on their color modifying ability, phenol oxidizing enzymes of the present invention can be used, for example, for pulp and paper bleaching, for bleaching the color of stains on fabric and for anti-dye transfer in detergent and textile applications. In one aspect of the present invention, the phenol oxidizing enzyme is able to modify the color in the absence of an enhancer. In another aspect of the present invention, the phenol oxidizing enzyme is able to modify the color in the presence of an enhancer.
The present invention is based upon the identification and characterization of a polynucleotide sequence (SEQ ID NO:1) encoding a phenol oxidizing enzyme obtainable from Stachybotrys and having the deduced amino acid sequence as shown in SEQ ID NO:2. The Stachybotrys genomic sequence is provided in SEQ ID NO:3. Accordingly, the present invention provides phenol oxidizing enzymes obtainable from Stachybotrys and comprising at least 65% identity, at least 70%, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2 as long as the enzyme is capable of modifying the color associated with dyes or colored compounds. In one embodiment, the phenol oxidizing enzyme has the amino acid sequence as shown in SEQ ID NO:2.
In another embodiment, the phenol oxidizing enzyme is obtainable from a Stachybotrys species including Stachybotrys parvispora, Stachybotrys chartarum; S. kampalensis; S. theobromae; S. bisbyi, S. cylindrospora, S. dichroa, S. oenanthes and S. nilagerica. In a preferred embodiment, the Stachybotrys includes Stachybotrys chartarum MUCL 38898 and S. chartarum MUCL 30782.
In yet another embodiment, the present invention provides an isolated polynucleotide encoding a phenol oxidizing enzyme obtainable from Stachybotrys wherein said polynucleotide comprises a nucleic acid sequence having at least 65% identity, at least 70%, at least 75% identity, at least 80%, at least 85%, at least 90% and at least 95% identity to SEQ ID NO:1 as long as the polynucleotide encodes a phenol oxidizing enzyme capable of modifying the color associated with dyes or colored compounds. The present invention also encompasses polynucleotide sequences that hybridize under conditions of high stringency to the polynucleotide shown in SEQ ID NO:1 or SEQ ID NO:3. The present invention also provides polynucleotides that encode the amino acid sequence as shown in SEQ ID NO:2. In a preferred embodiment, the polynucleotide has the nucleic acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:3. The present invention also provides expression vectors and host cells comprising polynucleotides of the present invention.
The present invention additionally relates to methods for producing a phenol oxidizing enzyme obtainable from Stachybotrys. Accordingly, the present invention provides a method for producing said enzyme comprising the steps of obtaining a host cell comprising a polynucleotide encoding said phenol oxidizing enzyme obtainable from Stachybotrys wherein said enzyme has at least 65% identity to the amino acid sequence disclosed in SEQ ID NO:2; growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme; and optionally recovering said phenol oxidizing enzyme produced. In one embodiment, the polynucleotide comprises the sequence as shown in SEQ ID NO:1. In another embodiment, the polynucleotide comprises the sequence as shown in SEQ ID NO: 3.
The present invention also provides a method for producing a host cell comprising a polynucleotide encoding a phenol oxidizing enzyme of the present invention comprising the steps of obtaining a polynucleotide encoding said phenol oxidizing enzyme obtainable from Stachybotrys and having at least 65% identity to the amino acid sequence disclosed in SEQ ID NO:2; introducing said polynucleotide into said host cell; and growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme. In another embodiment, the polynucleotide comprises the sequence as shown in SEQ ID NO: 3.
In one aspect of the present invention, the host cell comprising a polynucleotide encoding a phenol oxidizing enzyme includes filamentous fungus, yeast and bacteria. In one embodiment, the host cell is a filamentous fungus including Aspergillus species, Trichoderma species and Mucor species. In a preferred embodiment, the filamentous fungus host cell includes Aspergillus niger var. awamori and Trichoderma reseei. 
In another embodiment of the present invention, the host cell is a yeast which includes Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces and Yarrowia species. In yet a another embodiment, the Saccharomyces species is Saccharomyces cerevisiae. In an additional embodiment, the host cell is a gram positive bacteria, such as a Bacillus species, or a gram negative bacteria, such as an Escherichia species.
Also provided herein are detergent compositions comprising the amino acid having at least 65% identity, at least 70%, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2 as long as the enzyme is capable of modifying the color associated with dyes or colored compounds. In a preferred embodiment, the amino acid has the sequence as shown in SEQ ID NO: 2.
The present invention also encompasses methods for modifying the color associated with dyes or colored compounds which occur in stains on fabric, comprising the steps of contacting the fabric with a composition comprising an amino acid sequence having at least 65% identity, at least 70%, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2 as long as the enzyme is capable of modifying the color associated with dyes or colored compounds. In a preferred embodiment of the method, the amino acid has the sequence as shown in SEQ ID NO:2. In one aspect of the method, the pH optimum is between 5.0 and 11.0, in another aspect, the pH optimum is between 7 and 10.5 and in yet another aspect the pH optimum is between 8.0 and 10. In a further aspect of the method, the optimum temperature is between 20 and 60 degrees C. and in another aspect between 20 and 40 degrees C. The present invention also provides methods for preventing dye transfer in detergent and textile applications.